Detection of antigen receptor gene rearrangements in lymphoproliferative malignancies by fluorescent polymerase chain reaction.
Monoclonal rearrangements of antigen receptor genes in lymphoproliferative diseases are characterized by the specific sequence and the length of their junctional region, which can be used as markers of the proliferating clone. PCR techniques have greatly simplified routine detection of monoclonal rearrangements. But on the one hand, identification of the sequences requires sequencing methods and on the other hand, sizing of rearrangements by conventional analysis of PCR products on agarose or nondenaturing polyacrylamide gels may be uncertain. We have developed an approach based on amplification of rearranged IGH, TCRG and TCRD locus by fluorescent PCR associated to a computerized analysis of generated PCR products allowing their objective sizing. We tested this method on DNA samples from patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia, whose pattern of IGH and TCRG rearrangements had been previously identified by Southern blot techniques. TCRG-PCR assay allowed detection of 100% of rearranged samples. No false-negative results were found but a high rate (60%) of Southern-negative and PCR-positive samples were identified. TCRD PCR-assay detected VD1-JD1 or VD2-D2/3 rearrangements in both acute lymphoblastic leukemia and chronic lymphocytic leukemia samples. IGH PCR assay permitted detection of all known rearranged samples. The sensitivity of these three different PCR assays (1% leukemic cells) was equivalent to that of other published PCR protocols. These results show the validity and reliability of the fluorescent PCR method for routine detection of IGH, TCRG and TCRD rearrangements. Sizing of PCR products by computerized analysis was also validated. It provides additional information on rearrangement patterns in lymphoproliferative diseases, as clonal rearrangements can be recognized by their size. This can be of great interest in various circumstances, particularly for detection and follow-up of oligoclonality.
Kerlan-Candon
S
S
Laboratoire d'Immunologie, INSERM U291, Hopital Saint Eloi, CHU Montpellier, France.
Soua
Z
Z
Lefranc
M P
MP
Clot
J
J
Eliaou
J F
JF
D002648:Child; D005456:Fluorescent Dyes; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D015329:Gene Rearrangement, T-Lymphocyte; D016694:Gene Rearrangement, delta-Chain T-Cell Antigen Receptor; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000276:immunology; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*
D005456:Fluorescent Dyes; D007143:Immunoglobulin Heavy Chains; D016692:Receptors, Antigen, T-Cell, gamma-delta